The present invention is concerned with genetic engineering to effect expression of lipoproteins from vectors containing nucleic acid molecules encoding the lipoproteins. More particularly, the present invention relates to expression of a recombinant lipoprotein wherein the lipidation thereof is from expression of a first nucleic acid sequence and the protein thereof is from expression of a second nucleic acid sequence, the first and second nucleic acid sequences, which do not naturally occur together, being contiguous. The invention further relates to expression of such lipoproteins wherein the first nucleic acid sequence encodes a Borrelia lipoprotein leader sequence. The invention also relates to recombinant lipidated proteins expressed using the nucleic acid sequence encoding the OspA leader sequence, methods of making and using the same compositions thereof and methods of using the compositions. The invention additionally relates to nucleic acid sequences encoding the recombinant lipoproteins, vectors containing and/or expressing the sequences, methods for expressing the lipoproteins and methods for making the nucleic acid sequences and vectors; compositions employing the lipoproteins, including immunogenic or vaccine compositions, such compositions preferably having improved immunogenicity; and methods of using such compositions to elicit an immunological or protective response.
Throughout this specification, various documents are referred to in order to more fully describe the state of the art to which this invention pertains. These documents are each hereby incorporated herein by reference.
Lyme borreliosis is the most prevalent tick-borne disease in the United States as well as one of the most important tick-borne infectious diseases worldwide. The spirochete Borrelia burgdorferi is the causative agent for Lyme disease. Infection with B. burgdorferi produces local and systemic manifestations. Local symptoms that appear early after infection are a skin lesion at the site of the tick bite, termed erythema migrans. Weeks to months after infection, systemic manifestations that include rheumatic, cardiac and neurological symptoms appear. The early local phase of B. burgdorferi infection is easily treatable with antibiotics. However, the later systemic phases have proved to be more refractory to antibiotics.
Substantial effort has been directed toward the development of a vaccine for Lyme disease. Two distinct approaches have been used for vaccine development. One approach is to use a vaccine composed of whole inactivated spirochetes, as described by Johnson in U.S. Pat. No. 4,721,617. A whole inactivated vaccine has been shown to protect hamsters from challenge and has been licensed for use in dogs.
Due to the concerns about cross-reactive antigens within a whole cell preparation, human vaccine research has focused on the identification and development of non-cross-reactive protective antigens expressed by B. burgdorferi. Several candidate antigens have been identified to date. Much of this effort has focused on the most abundant outer surface protein of B. burgdorferi, namely outer surface protein A (OspA), as described in published PCT patent application WO 92/14488, assigned to the assignee hereof. Several versions of this protein have been shown to induce protective immunity in mouse, hamster and dog challenge studies. Clinical trials in humans have shown the formulations of OspA to be safe and immunogenic in humans [Keller et al., JAMA (1994) 271:1764-1768]. Indeed, one formulation containing recombinant lipidated OspA as described in the aforementioned WO 92/14488, is now undergoing Phase III safety/efficacy trials in humans.
While OspA is expressed in the vast majority of clinical isolates of B. burgdorferi from North America, a different picture has emerged from examination of the clinical Borrelia isolates in Europe. In Europe, Lyme disease is caused by three genospecies of Borrelia, namely B. burgdorferi, B. garinii and B. afzelli. In approximately half of the European isolates, OspA is not the most abundant outer surface protein. A second outer surface protein C (OspC) is the major surface antigen found on these spirochetes. In fact, a number of European clinical isolates that do not express OspA have been identified. Immunization of gerbils and mice with purified recombinant OspC produces protective immunity to B. burgdorferi strains expressing the homologous OspC protein [V. Preac-Mursic et al., INFECTION (1992) 20:342-349; W. S. Probert et al., INFECTION AND IMMUNITY (1994) 62:1920-1926]. The OspC protein is currently being considered as a possible component of a second generation Lyme vaccine formulation.
Recombinant proteins are promising vaccine or immunogenic composition candidates, because they can be produced at high yield and purity and manipulated to maximize desirable activities and minimize undesirable ones. However, because they can be poorly immunogenic, methods to enhance the immune response to recombinant proteins are important in the development of vaccines or immunogenic compositions.
A very promising immune stimulator is the lipid moiety N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine, abbreviated Pam3Cys. This moiety is found at the amino terminus of the bacterial lipoproteins which are synthesized with a signal sequence that specifies lipid attachment and cleavage by signal peptidase II. Synthetic peptides that by themselves are not immunogenic induce a strong antibody response when covalently coupled to Pam3Cys [Bessler et al., Research Immunology (1992) 143:548-552].
In addition to an antibody response, one often needs to induce a cellular immune response, particularly cytoxic T lymphocytes (CTLs). Pam3Cys-coupled synthetic peptides are extremely potent inducers of CTLs, but no one has yet reported CTL induction by large recombinant lipoproteins.
The nucleic acid sequence and encoded amino acid sequence for OspA are known for several B. burgdorferi clinical isolates and is described, for example, in published PCT application WO 90/04411 (Symbicom AB) for B31 strain of B. burgdorferi and in Johnson et al., Infect. Immun. 60:1845-1853 for a comparison of the ospA operons of three B. burgdorferi isolates of different geographic origins, namely B31, ACA1 and Ip90.
As described in WO 90/04411, an analysis of the DNA sequence for the B31 strain shows that the OspA is encoded by an open reading frame of 819 nucleotides starting at position 151 of the DNA sequence and terminating at position 970 of the DNA sequence (see FIG. 1 therein). The first sixteen amino acid residues of OspA constitute a hydrophobic signal sequence of OspA. The primary translation product of the full length B. burgdorferi gene contains a hydrophobic N-terminal signal sequence which is a substrate for the attachment of a diacyl glycerol to the sulfhydryl side chain of the adjacent cysteine residue. Following this attachment, cleavage by signal peptidase II and the attachment of a third fatty acid to the N-terminus occurs. The complete lipid moiety is termed Pam3Cys. It has been shown that lipidation of OspA is necessary for immunogenicity, since OspA lipoprotein with an N-terminal Pam3Cys moiety stimulated a strong antibody response, while ospA lacking the attached lipid did not induce any detectable antibodies [Erdile et al., Infect. Immun., (1993), 61:81-90].
Published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA sequence of the ospC gene of B. burgdorferi strain Pko and the OspC (termed pC in this reference) protein encoded thereby of 22 kDa molecular weight. This sequence reveals that OspC is a lipoprotein that employs a signal sequence similar to that used for OspA. Based on the findings regarding OspA, one might expect that lipidation of recombinant OspC would be useful to enhance its immunogenicity; but, as discussed below, the applicants experienced difficulties in obtaining detectable expression of recombinant OspC.
U.S. Pat. No. 4,624,926 to Inouye relates to plasmid cloning vectors, including a DNA sequence coding for a desired polypeptide linked with one or more functional fragments derived from an outer membrane lipoprotein gene of a gram negative bacterium. The polypeptide expressed by the transformed E. coli host cells comprises the signal peptide of the lipoprotein, followed by the first eight amino acid residues of the lipoprotein, which in turn are followed by the amino acid sequence of the desired protein. The signal peptide may then be translocated naturally across the cytoplasmic membrane and the first eight amino acids of the lipoprotein may then be processed further and inserted into the outer membrane of the cell in a manner analogous to the normal insertion of the lipoprotein into the outer membrane. Immunogenicity of the expressed proteins was not demonstrated. Moreover, Inouye was not at all concerned with recombinant lipidation, particularly to enhance immunogenicity.
Published international patent application WO91/09952 describes plasmids for expressing lipidated proteins. Such plasmids involve a DNA sequence encloding a lipoprotein signal peptide linked to the codons for one of the xcex2-turn tetrapeptides QANY or IEGR, which in turn is linked to the DNA sequence encoding the desired protein. Again, immunogenicity of the expressed proteins was not demonstrated.
Streptoccus pneumoniae causes more fatal infections world-wide than almost any other pathogen. In the U.S.A., deaths caused by S. pneumoniae rival in numbers those caused by AIDS. Most fatal pneumoccal infections in the U.S.A. occur in individuals over 65 years of age, in whom S. pneumoniae is the most common cause of community-acquired pneumonia. In the developed world, most pneumococcal deaths occur in the elderly, or in immunodeficient patents including those with sickle cell disease. In the less-developed areas of the world, pneumococcal infection is one of the largest causes of death among children less than 5 years of age. The increase in the frequency of multiple antibiotic resistance among pneumococci and the prohibitive cost of drug treatment in poor countries make the present prospect for control of pneumococcal disease problematical.
The reservoir of pneumococci that infect man is maintained primarily via nasopharyngeal human carriage. Humans acquire pneumococci first through aerosols or by direct contact. Pneumococci first colonize the upper airways and can remain in nasal mucosa for weeks or months. As many as 50% or more of young children and the elderly are colonized. In most cases, this colonization results in no apparent infection. In some individuals, however, the organism carried in the nasopharynx can give rise to symptomatic sinusitis of middle ear infection. If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia. Infections at these sites generally shed some pneumococci into the blood where they can lead to sepsis, especially if they continue to be shed in large numbers from the original focus of infection. Pneumococci in the blood can reach the brain where they can cause menigitis. Although pneumococcal meningitis is less common than other infections caused by these bacteria, it is particularly devastating; some 10% of patients die and greater than 50% of the remainder have life-long neurological sequelae.
In elderly adults, the present 23-valent capsular polysaccharide vaccine is about 60% effective against invasive pneumococcal disease with strains of the capsular types included in the vaccine. The 23-valent vaccine is not effective in children less than 2 years of age because of their inability to make adequate responses to most polysaccharides. Improved vaccines that can protect children and adults against invasive infections with pneumococci would help reduce some of the most deleterious aspects of this disease.
The S. pneumoniae cell surface protein PspA has been demonstrated to be a virulence factor and a protective antigen. In published international patent application WO 92/14488, there are described the DNA sequences for the pspA gene from S. pneumoniae Rx1, the production of a truncated form of PspA by genetic engineering, and the demonstration that such truncated form of PspA confers protection in mice to challenge with live pneumococci.
In an effort to develop a vaccine or immunogenic composition based on PspA, PspA has been recombinantly expressed in E. coli. It has been found that in order to efficiently express PspA, it is useful to truncate the mature PspA molecule of the Rx1 strain from its normal length of 589 amino acids to that of 314 amino acids comprising amino acids 1 to 314. This region of the PspA molecule contains most, if not all, of the protective epitopes of PspA. However, immunogenicity and protection studies in mice have demonstrated that the truncated recombinant form of PspA is not immunogenic in naive mice. Thus, it would be useful to improve the immunogenicity of recombinant PspA and fragments thereof.
Many bacterial and viral pathogens, such as S. pneumoniae and Helicobacter pylori, and HIV, herpes and papilloma viruses gain entry through mucosal surfaces. The principal determinant of specific immunity at mucosal surfaces is secretory IgA (S-IgA) which is physiologically and functionally separate from the components of the circulatory immune system. Mucosal S-IgA responses are predominantly generated by the common mucosal immune system (CMIS) [Mestecky, J. Clin. Immunol. (1987), 7:265-276], in which immunogens are taken up by specialized lympho-epithelial structures collectively referred to as mucosa-associated lymphoid tissue (MALT). The term common mucosal immune system referes to the fact that immunization at any mucosal site can elicit an immune response at all other mucosal sites. Thus, immunization in the gut can elicit mucosal immunity in the upper airways and vice versa. Further, it is important to note that oral immunization can induce an antigen-specific IgG response in the systemic compartment in addition to mucosal IgA antibodies [McGhee, J. R. et al., (1993), Infect. Agents and Disease 2:55-73].
Most soluble and non-replicating antigens are poor mucosal immunogens, especially by the peroral route, probably because they are degraded by digestive enzymes and have little or no tropism for the gut associated lymphoid tissue (GALT). Thus, a method for producing effective mucosal immunogens, and vaccines and immunogenic compositins containing them, would be desirable.
Of particular interest is H. pylori, the spiral bacterium which selectively colonizes human gastric mucin-secreting cells and is the causative agent in most cases of nonerosive gastritis in humans. Recent research indicates that H. pylori, which has a high urease activity, is responsible for most peptic ulcers as well as many gastric cancers. Many studies have suggested that urease, a complex of the products of the ureA and ureB genes, may be a protective antigen, However, until now it has not been known how to produce a sufficient mucosal immune response to urease.
Antigens, such as OspC, PspA, UreA, UreB or immunogenic fragments thereof, stimulate an immune response when administered to a host. Such antigens, especially when recombinantly produced, may elicit a stronger response when administered in conjunction with an adjuvant. Currently, alum is the only adjuvant licensed for human use, although hundreds of experimental adjuvants such as cholera toxin B are being tested. However, these adjuvants have deficiencies. For instance, while cholera toxin B is not toxic in the sense of causing cholera, there is general unease about administering a toxin associated with a disease as harmful as cholera, especially if there is even the most remote chance of minor impurity.
Thus, it would be desirable to enhance the immunogenicity of antigens, by methods other than the use of an adjuvant, especially in monovalent preparations; and, in multivalent preparations, to have the ability to employ such a means for enhanced immunogenicity with an adjuvant, so as to obtain an even greater immunological response.
As to expression of recombinant proteins, it is expected that the skilled artisan is familiar with the various vector systems available for such expression, e.g., bacteria such as E. coli and the like.
It is believed that heretofore the art has not taught or suggested expression of a recombinant lipoprotein wherein the lipidation thereof is from expression of a first nucleic acid sequence, the protein thereof is from expression of a second nucleic acid sequence, the first and second sequences, which do not naturally occur together, being contiguous, especially such a lipoprotein wherein the first sequence a Borrelia lipoprotein leader sequence, preferably an OspA leader sequence, and even more preferably wherein the first sequence encodes an OspA leader sequence and the second sequence encodes OspC, PspA, UreA, UreB, or an immunogenic fragment thereof; or genes containing such sequences; or vectors containing such sequences; or methods for such expression; or such recombinant lipoproteins; or compositions containing such recombinant lipoproteins; or methods for using such compositions; or methods for enhancing the immunogenicity of a protein by lipidation from a nucleic acid sequence not naturally occurring with the nucleic acid sequence encoding the protein portion of the lipoprotein.
It is an object of the invention to provide a recombinant lipoprotein wherein the lipidation thereof is from expression of a first nucleic acid sequence and the protein portion thereof is from expression of a second nucleic acid sequence and the first and second sequences do not naturally occur together; especially such a lipoprotein wherein the first sequence encodes a Borrelia lipoprotein leader sequence, preferably an ospA leader sequence, and more preferably wherein the second sequence encodes a protein portion comprising OspC, PspA, UreA, UreB, or an immunogenic fragment thereof.
It is another object of the invention to provide expression of genes and/or sequences encoding such a recombinant lipoprotein, vectors therefor and methods for effecting such expression.
It is a further object of the invention to provide immunogenic compositions, including vaccines, containing the recombinant lipoproteins and/or vectors for expression thereof.
It has surprisingly been found that an immunogenic recombinant lipidated protein, preferably OspC or a portion thereof, can be expressed from a vector system, preferably E. coli, without the toxicity to the vector system evident when the native lipoprotein signal sequence encoding region is present. This result has been achieved by replacing the nucleotide sequence encoding the native leader or signal sequence of a lipoprotein with the nucleotide sequence encoding a leader or signal of another lipoprotein, preferably of a Borrelia lipoprotein, and more preferably the OspA leader or signal sequence. Proteins not naturally lipidated, such as PspA, UreA, and UreB, may be expressed as recombinant lipidated proteins as well, by fusing lipoprotein signal sequence to the first amino acid of the desired protein. These recombinant lipidated proteins have been shown to elicit an immune response, including a mucosal immune response.
It is surprising that fusion of DNA encoding a lipoprotein leader sequence directly to the DNA encoding a protein, without any intervening nucleotide sequences, can lead to expression of an immunogenic recombinant lipoprotein in significant quantities without the toxicity evident with the native leader sequence, because previous attempts to express recombinant lipidated proteins have been unsuccessful. For example, Fuchs et al. report that recombinantly formed OspC (referred to as pC in this reference) with its native leader protein was only weakly expressed in E. coli [Mol. Microbiology (1992) 6(4):503-509]. Applicants, in addition to Fuchs, attempted to obtain lipidated recombinant OspC by expression of the OspC-encoding sequence in E. coli using the pET vector system described in the aforementioned WO 92/14488 for the expression of OspA and using the pDS12 plasmid systems described. However, OspC was barely detectable by immunoblotting of cell extracts using these systems to express OspC.
Further, as discussed supra, it was believed that an additional nucleotide sequence, preferably one encoding a peptide sequence forming a xcex2-turn, was necessary for expression of recombinant lipoproteins, and the immunogenicity of recombinant lipoproteins previously expressed had not been demonstrated.
The procedure of the present invention, therefore, enables large quantities of pure recombinant, immunogenic lipidated proteins, e.g., OspC, PspA, UreA, UreB and portions thereof, to be obtained, which has not heretofore been possible. The recombinantly-formed lipidated proteins provided herein are significantly more immunogenic than a non-lipidated recombinant analog.
The present invention, it is believed, represents the first instance of effecting expression of a heterologous lipidated protein using a non-native, preferably Borrelia and more preferably the OspA leader sequence. The invention, therefore, includes the use of non-native, preferably Borrelia and more preferably the OspA leader sequence to express proteins heterologous to the leader sequence.
Accordingly, in one embodiment, the present invention provides an isolated hybrid nucleic acid molecule, preferably DNA, comprising a first nucleic acid sequence encoding the signal sequence preferably of an OspA protein of a Borrelia species, coupled in translational open reading frame relationship with a second nucleic acid sequence encoding a mature protein heterologous to the signal sequence, preferably to OspC or PspA. More preferably, the first and second sequences are contiguous when the mature protein is naturally lipidated, and separated by one codon coding for one amino acid, preferably cysteine, when the mature protein is not naturally lipidated.
The mature protein encoded by the second nucleic acid sequence generally is a lipoprotein, preferably an antigenic lipoprotein,an more preferably is the mature OspC lipoprotein of a Borrelia species, preferably a strain of B. burgdorferi, more preferably a strain of B. burgdorferi selected from the OspC sub-type families. In another preferred embodiment, the mature protein is the mature PspA protein, or an immunogenic fragment thereof, of a strain of S. pneumoniae. In yet another preferred embodiment, the mature protein is UreA or UreB protein of a strain of H. pylori. Similarly, the signal sequence of the OspA protein of a Borrelia strain encoded by the first nucleic acid sequence preferably is that of a strain of B. burgdorferi, more preferably a strain of B. burgdorferi selected from the B31, ACA1 and Ip90 families of strains.
The hybrid gene provided herein may be assembled into an expression vector, preferably under the control of a suitable promoter for expression of the mature lipoprotein, in accordance with a further aspect of the invention, which, in a suitable host organism, such as E. coli, causes initial translation of a chimeric molecule comprising the leader sequence and the desired heterologous protein in lipidated form, followed by cleavage of the chimeric molecule by signal peptidase II and attachment of lipid moieties to the new terminus of the protein, whereby the mature lipoprotein is expressed in the host organism.
The present invention provides, for the first time, a hybrid nucleic acid molecule which permits the production of recombinant lipidated protein, e.g., recombinant lipidated OspC of a Borrelia species, recombinant lipidated PspA of a strain of S. pneumoniae or recombinant lipidated UreA or UreB of a strain of H. pylori, to be obtained. Accordingly, in a further aspect of this invention, there is provided a hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a lipoprotein, preferably an OspC lipoprotein of a Borrelia species, more preferably a strain of B. burgdorferi, still more preferably a strain of B. burgdorferi selected from the OspC sub-type families; or encoding a PspA lipoprotein of a strain of S. pneumoniae or immunogenic fragment thereof; or encoding a UreA or UreB lipoprotein of a strain of H. pylori; and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterologous to the protein encoded by the first nucleic acid sequence and coupled in translational open reading frame relationship with said first nucleic acid sequence, preferably encoding the signal sequence of an OspA protein of a Borrelia species.
As described above, the hybrid gene can be assembled into an expression vector under the control of a suitable promoter for expression of the lipoprotein, which, in a suitable host organism, such as E. coli, causes expression of the lipoprotein from the host organism.
It has also surprisingly been found that enhanced immunogenicity can be obtained by a recombinant lipoprotein when the lipoprotein is expressed by a hybrid or chimeric gene comprising a first nucleic acid sequence encoding a leader or signal sequence and a second nucleic acid sequence encoding the protein portion of the lipoprotein, wherein the first and second sequences do not naturally occur together.
Accordingly, the present invention also provides a recombinant lipoprotein expressed by a hybrid or chimeric gene comprising a first nucleic acid sequence encoding a leader or signal sequence contiguous with a second nucleic acid sequence encoding a protein portion of the lipoprotein, and the first and second sequences do not naturally occur together. The first and second sequences are preferably coupled in a translational open reading frame relationship. The first sequence can encode a leader sequence of a Borrelia lipoprotein, preferably the leader sequence of OspA; and the second sequence can encode a protein comprising an antigen, preferably OspC, PspA, UreA, UreB or an immunogenic fragment thereof. The first and second sequences can be present in a gene; and the gene and/or the first and second sequences can be in a suitable vector for expression.
The vector can be a nucleic acid in the form of, e.g., plasmids, bacteriophages and integrated DNA, in a bacteria, most preferably one used for expression, e.g. E. coli, Bacillus subtilis, Salmonella, Staphylocoocus, Streptococcus, etc., or one used as a live vector, e.g. Lactobacillus, Mycobacterium, Salmonella, Streptococcus, etc. When an expression host is used the recombinant lipoprotein can be obtained by harvesting product expressed in vitro; e.g., by isolating the recombinant lipoprotein from a bacterial extract. The gene can preferably be under the control of and therefore operably linked to a suitable promoter; and the promoter can either be endogenous to the vector, or be inserted into the vector with the gene.
The invention further provides vectors containing the nucleic acid encoding the recombinant lipoprotein and methods for obtaining the recombinant lipoproteins and methods for preparing the vector.
As mentioned, the recombinant lipoprotein can have enhanced immunogenicity. Thus, additional embodiments of the invention provide immunogenic or vaccine compositions for inducing an immunological response, comprising the isolated recombinant lipoprotein, or a suitable vector for in vivo expression thereof, or both, and a suitable carrier, as well as to methods for eliciting an immunological or protective response comprising administering to a host the isolated recombinant lipoprotein, the vector expressing the recombinant lipoprotein, or a composition containing the recombinant lipoprotein or vector, in an amount sufficient to elicit the response.
Documents cited in this disclosure, including the above-referenced applications, provide typical additional ingredients for such compositions, such that undue experimentation is not required by the skilled artisan to formulate a composition from this disclosure. Such compositions should preferably contain a quantity of the recombinant lipoprotein or vector expressing such sufficient to elicit a suitable response. Such a quantity of recombinant lipoproprotein or vector can be based upon known amounts of antigens administered. For instance, if there is a known amount for administration of an antigen corresponding to the second sequence expressed for the inventive recombinant lipoprotein, the quantity of recombinant lipoprotein can be scaled to about that known amount, and the amount of vector can be such as to produce a sufficient number of colony forming units (cfu) so as to result in in vivo expression of the recombinant lipoprotein in about that known amount. Likewise, the quantity of recombinant lipoprotein can be based upon amounts of antigen administered to animals in the examples below and in the documents cited herein, without undue experimentation.
The present invention also includes, in other aspects, processes for the production of a recombinant lipoprotein, by assembly of an expression vector, expression of the lipoprotein from a host organism containing the expression vector, and optionally isolating and/or purifying the expressed lipoprotein. The isolating/purifying can be so as to obtain recombinant lipoprotein free from impurities such as lipopolysaccharides and other bacterial proteins. The present invention further includes immunogenic compositions, such as vaccines, containing the recombinant lipoprotein as well as methods for inducing an immunological response.